Large Extracellular Spaces Research Articles

Possible maternal inheritance of breast muscle morphology in turkeys at sixteen weeks of age

During the study of inheritance of breast muscle morphology of turkeys during embryonic development and posthatch in two experiments, interesting results were obtained at 16 wk of age. In experiment 1, an experimental line (F) selected long term for increased 16-wk BW was crossed reciprocally with a commercial sire line (B). Samples of pectoralis major muscle were obtained from three males and three females of each genetic group. The F line was reciprocally crossed with a randombred control line (RBC2) in experiment 2, and p. major muscle samples were taken from 10 males and 10 females of each genetic group. The RBC2 line was the base population for the F line. The muscle samples were obtained in a manner to avoid contraction. After fixing, the muscle samples were stained with hematoxylin and eosin to view muscle morphology. Distinct morphological types were observed in the muscle samples for the B, F, and RBC2 lines. The pectoralis major muscle from the B line was, in general, characterized by large fibers with a well-defined extracellular matrix (perimysial and endomysial spaces). Similar to the B line, representative samples of the RBC2 line had a well-defined extracellular matrix but muscle fiber size was not as large as that of the B line. Representative samples of the F line were characterized by a greatly reduced extracellular matrix with the muscle fibers not well defined. For the reciprocal crosses in both experiments, representative samples indicated the offspring had breast muscle morphology similar to that of the female parent, suggesting maternal inheritance. There was no sex effect on breast muscle morphology in either experiment. In order to study the consistency of the results, muscle morphology of representative sections of muscle were subjectively rated by four individuals. The ratings ranged from 1 (little extracellar matrix and indistinct muscle fibers) to 5 (large extracellular space and distinct muscle fibers). Ratings of 2 to 4 were intermediate to these extremes. In experiment 1, ratings of the pure B line and the F sire × B dam cross were similar and higher than that of the pure F line and B sire × F dam cross, which did not differ in average rating. The results for experiment 2 were similar to those for experiment 1 in that the average ratings of the reciprocal crosses were similar to that of the female parental line. The results from the ratings support maternal inheritance of breast muscle morphology.

Ephaptic Interactions in the Mammalian Olfactory System

Ephaptic coupling refers to interactions between neurons mediated by current flow through the extracellular space. Ephaptic interactions between axons are considered negligible, because of the relatively large extracellular space and the layers of myelin that separate most axons. By contrast, olfactory nerve axons are unmyelinated and arranged in tightly packed bundles, features that may enhance ephaptic coupling. We tested the hypothesis that ephaptic interactions occur in the mammalian olfactory nerve with the use of a computational approach. Numerical solutions of models of axon fascicles show that significant ephaptic interactions occur for a range of physiologically relevant parameters. An action potential in a single axon can evoke action potentials in all other axons in the fascicle. Ephaptic interactions can also lead to synchronized firing of independently stimulated axons. Our findings suggest that ephaptic interactions may be significant determinants of the olfactory code and that such interactions may occur in other, similarly organized axonal or dendritic bundles.

Interstitial cells of Cajal: primary targets of enteric motor innervation.

For many years morphologists have noted the close relationship between interstitial cells of Cajal (ICC) and nerve fibers within the tunica muscularis of gastrointestinal (GI) organs. These observations led to speculations about a role for ICC in mediating neural inputs to the GI tract. Immunohistochemical and functional studies demonstrated the presence of receptors for the neurotransmitters utilized by enteric motor neurons, and changes in second messengers in ICC after field stimulation of intrinsic enteric neurons showed that ICC were functionally innervated in GI muscles. Recent double labeling experiments have shown that both excitatory and inhibitory enteric motor neurons are closely associated with ICC in the deep muscular plexus (IC-DMP) of the small intestine and intramuscular ICC (IC-IM) of the proximal and distal GI tract. Enteric motor neurons form synaptic-like structures with IC-IM and IC-DMP. Far fewer close contacts are found between enteric motor neurons and smooth muscle cells. Experiments on W/W(V) mutants that lack IC-IM in the stomach, lower esophageal sphincter, and pylorus have shown that these ICC are critical components of the neuromuscular junction. Cholinergic excitatory and nitrergic inhibitory neurotransmission are severely decreased in tissues lacking IC-IM, yet there is no loss of cholinergic or nitrergic neurons in W/W(V) mutants. These data suggest that either the post-junctional mechanisms responsible for receiving and transducing neurotransmitter signals are specifically expressed by ICC, or that the large extracellular spaces typically between nerve terminals and smooth muscle cells may not allow effective concentrations of neurotransmitters to reach receptors expressed by smooth muscle cells. These findings indicate an important role for certain classes of ICC in enteric neurotransmission and predict that loss of ICC in human motor disturbances may significantly compromise neural regulation of GI motility.

Diffusion-weighted echo planar imaging of the normal human cervical spinal cord.

We obtained diffusion-weighted echo planar images of the human cervical cord in vivo and correlated them with histopathologic findings. Images were obtained in 17 healthy volunteers using a 1.5 T clinical MR unit. When motion-probing gradients were added perpendicular to the long axis of the cord, the white matter was hyperintense because of anisotropic diffusion. However, the gracile fasciculus was hypointense probably due to the small diameter of neural fibers and the large extracellular space.

PHARMACOLOGIC DOSES OF INTERLEUKIN 8 SUPPOSITORIES INDUCE FOLLICULAR MATURATION IN RABBITS

Interleukin 8 (IL-8) is a neutrophil chemoattractant/activating factor that plays a role in the ovarian physiology leads to investigate the effects of IL-8 on follicular maturation. Experiments were conducted using suppositories containing 100ng, 200ng, 400ng IL-8, 500μl Witepsol-base (control), human menopausal gonadotropin (im) and conjugate of fluorescein isithiocyanate-labelled IL-8. The levels of IL-8 in ovarian fluid were also measured. Histology of ovaries treated with 200ng IL-8 showed large antral follicles filled with follicular fluid. The theca layer was divided into an interna and an externa with large extracellular spaces. The granulosa cells were loosened and appeared to be detaching from the granulosa layer. Neutrophils were localized predominantly in the theca and medulla (P<0.0001, P<0.004), and relative collagen concentration was significantly decreased in ovaries of 200ng IL-8 (P<0.0001) compared with controls. The IL-8 was detected in ovarian fluid after 6h (P<0.0001), 12h (P<0.001), and 18h (P<0.01) compared with 0h. Fluorescein isithiocyanate-labelled IL-8 conjugate was seen in the follicular wall and endometrium. We conclude that pharmacological dosage of exogenous IL-8 exerts an effect on follicular maturation through granulocyte chemotaxis and activation.

Reconstruction of trabecular tissue from human trabecular cells as a multicellular spheroid.

To understand the development of the trabecular meshwork of the eye, floating cellular aggregates (multicellular spheroids) were formed from human trabecular cells in a non-adherent environment of culture and incubated for up to one month. Dissociated trabecular cells formed multicellular spheroids within one day in the non-adherent environment, and apoptosis continued to occur in the spheroids which had been initially filled with cells. The final structure after one month appeared as a meshwork of cells with large extracellular spaces. Epidermal and basic fibroblast growth factor (EGF and bFGF) protected trabecular cells in the spheroids from apoptosis and, as a result, kept the spheroids filled with cells even after one month. In the absence of excess EGF or bFGF, the multicellular spheroids grown in vitro from human trabecular cells mimicked the mesh-like structure of normal trabecular tissue. In contrast, under an excess of these growth factors, spheroids of high cellularity, resembling the abnormal trabecular tissues of patients with congenital glaucoma, were formed.

Development of the anterior commissure in the opossum: Midline extracellular space and glia coincide with early axon decussation

While the anterior commissure has been shown to be an important route of information transfer in the forebrain, relatively little is known about its anatomical development. Glial substrates and extracellular spaces have been associated with the maturation of other large-fiber tracts, such as the corpus callosum and retinofugal pathway. The present study examined early stages in the maturation of the commissure in the gray short-tailed opossum, Monodelphis domestica. Monodelphis offspring are born after a short 14-day gestation, and, unlike in rats and mice, the anterior commissure develops entirely during the postnatal period. A number of techniques were employed: the carbocyanine dye Dil was used to label early axons in the region, semithin plastic sections were used to examine the extracellular environment of the developing commissure, and immunocytochemistry for glial fibrillary acidic protein (GFAP) was used to characterize glial components. Results suggest that the first commissural fibers that cross the midline pass through a region of large extracellular spaces and may use GFAP-immunoreactive cells and processes as guides during their midline decussation.

Biophysical aspects of meat tenderness

Biophysical aspects of meat tenderness is reviewed, where the meat structural origin of variation in meat tenderness is tried to be elucidated. Processes, such as rigor development and ageing, known to influence the properties of the structural components, is covered, and variables that influence those processes, such as chilling, electrical stimulation and stress ante-mortem, are discussed. Meat tenderness can be evaluated both by sensory and instrumental methods. The relationships between mechanical and sensory assessments tend to be non-linear, which can be due to non-linearity in the sensory evaluation and that muscle fibre orientation is easier to control in instrumental than in sensory evaluation. Structural changes of the meat occuring during rigor development are both longitudinal and lateral contraction of the myofibrillar mass. Other structural events, based on the proteolytic action, are the loosing up of the myofibrils held together laterally, weakening of the myofibrillar length and myofibril fragmentation. Using instrumental recordings of meat toughness (Warner-Bratzler (W-B) peak force), it decreases significantly with degree of contraction, when raw, but the reverse is found, when meat is cooked above 60 °C. A structural explanation to this behaviour is suggested to be the following. When meat is raw the lateral contraction of the meat fiber increases with shorter sarcomeres, giving rise to a larger viscous component and hence a lower W-B peak force. On heating, however, with a larger extracellular space, when shortened, there is more room for the connective tissue to contract without being restricted by the myofibrillar mass. This in turn gives a higher number of fibers per unit cross-sectional area, hence a larger elastic modulus and a higher W-B peak force, when cooked. When chilling of muscle during rigor both warm- and cold-shortening occur. Minimal shortening region is for beef M. longissimus dorsi (LD) 10–15 °C and for M. semimembranosus (SM) 7–13 °C. For the SM muscle there is a high correlation between percentage shortening and ultimate tenderness both in the warm- and cold-shortening region. But for the LD muscle this is only the case in the cold-shortening region. This observation suggests that the LD muscle is a more enzymatically active muscle than SM. The influence of low-voltage electrical stimulation (ES) was followed in the cold-shortening region for muscles LD and SM. A significant effect on tenderness 15 days post-mortem was only observed for LD at 1 °C and 4 °C, but not for SM. It was suggested that enhanced proteolysis could be the reason for the improved tenderness on ES of LD, as colds-hortening was not prevented by ES. Long-term and short-term stress ante-mortem can give rise to DFD (dark, firm and dry)-and PSE (pale, soft and exudative)-meat, respectively. DFD-meat (pH u > 6.0 in LD) has relatively short sarcomere lengths, but still it is swollen laterally and has consequently a small extracellular space. Therefore DFD-meat usually is tender. PSE-meat has a large variation in sarcomere length. The long sarcomeres of PSE-meat is suggested to be caused by reduced shortening, due to the denaturation of the sarcoplasmic proteins during rigor. The short sarcomeres can be caused by a higher percentage of rigor development in the warm-shortening region and that the denaturation of the myosin heads cause both longitudinal and lateral contraction of the myofibrillar mass. There is also a large variation in tenderness of PSE-meat, but it has been found that this variation is positively correlated to the sarcomere length (r = 0.52**), as has been shown for the other variables that governs the rigor process.

Cranial meninges of goldfish: age-related changes in morphology of meningeal cells and accumulation of surfactant-like multilamellar bodies.

In the optic tectum of goldfish, the outer, middle and inner layers of the endomeninx were evident in animals ranging in age from 1 month to several years. The outer layer in young animals consisted of closely overlapping cells with intertwined processes, whereas in the older animals it contained large extracellular spaces. The intermediate layer cells were always arranged in a single continuous layer, but in young animals they overlapped extensively with one another toward their edges whereas in the oldest animals they became extremely flat and non-overlapping. The inner layer included an outer tier of cells with their bases adhering to the intermediate layer, and an inner tier of cells detached from both the intermediate layer and the basal lamina overlying the brain parenchyma. Inner layer cells contained many large vacuoles that were in continuity with the extracellular space. With age, the extracellular space and the vacuolar system expanded, and the inner layer evolved into a meshwork of attenuated cytoplasmic processes embedded in the granular extracellular matrix. Another age-related feature was the accumulation adjacent to the basal lamina of uniform disc-shaped membranous structures, resembling multilamellar bodies of lung surfactant. These "disc bodies" were apparently generated by the coalescence of vesicles formed at the surface of the inner layer cells, possibly as a by-product of protein secretion by these cells.

Ultrastructure of calcitonin gene-related peptide-immunoreactive, unmyelinated afferents to the cat carotid body: A case of volume transmission

To relate the ultrastructure of unmyelinated afferents to the cat carotid body with the known electrophysiological properties of cat chemosensory C-fibers, we took advantage of the fact that the calcitonin gene-related peptide is exclusively present in a population of sparsely branched afferents to the carotid body. They have a morphology identical to the afferents originating from carotid sinus nerve unmyelinated axons. Immunoreactive axons were stained using pre-embedding protocols and horseradish peroxidase-labeled secondary antibody. Labeling was present only in unmyelinated axons and boutons distributed in the interstitial and parenchymal tissue. The varicosities had an average diameter of 0.7 micron, and contained both small, clear vesicles and larger dense-core vesicles. No labeled axons were ever seen to contact glomus cells, but could be observed as close as 0.2 micron to a glomus cell, always with an interposed glial process. With a very sensitive protocol, that used tungstate-stabilized tetramethylbenzidine as the chromogen, amorphous deposits of reaction product were often detected in the extracellular space around a labeled bouton. We interpret these findings as indicating that the reciprocal chemical transmission between the oxygen-sensitive glomus cells and the unmyelinated afferents takes place through non-synaptic transmission, via the rather large extracellular space of the carotid body. In addition, the larger distances between glomus cells and unmyelinated afferents could explain the lowered sensitivity and sluggishness of chemosensory C-fibers, compared to the A-fibers.

Extracellular space parameters in the rat neocortex and subcortical white matter during postnatal development determined by diffusion analysis

Extracellular space volume fraction, tortuosity and nonspecific uptake of tetramethylam monium—three diffusion parameters of brain tissue—were measured in gray matter of the somatosensory neocortex and subcortical white matter of the rat during postnatal development. The three parameters were determined from concentration-time profiles of tetramethylammonium in postnatal days 2–120 in vivo. Tetramethylammonium concentration was measured with ion-selective microelectrodes positioned 130–200 μm from an iontophoretic source. Data were correlated with cytoarchitectonic structure and average thickness of the regions in 0–90-day-old rats using rapidly frozen tissue. Extracellular space volume fraction was largest in the newborn rats and diminished with age. In two-to three-day-old animals, volume fraction (mean ± S.E.) was0.36 ± 0.04 in layers III and IV,0.38 ± 0.02 in layer V,0.41 ± 0.01 in layer VI and0.46 ± 0.01 in white matter. The earliest decrease in volume fraction was found in layers V and VI at postnatal days 6–7 followed by a decrease in layer III and IV at postnatal days 8–9 and in white matter at postnatal days 10–11. A further dramatic reduction in volume fraction occurred in all cortical layers and especially in the white matter between postnatal days 10 and 21. There was no further decrease in volume fraction between postnatal day 21 and adults (90–120 days old). The adult volume fraction values were: layer II,0.19 ± 0.002; III,0.20 ± 0.004; IV,0.21 ± 0.003; V,0.22 ± 0.003; VI,0.23 ± 0.007; white matter,0.20 ± 0.008. Values of tortuosity ranged between 1.51 and 1.65, nonspecific cellular uptake varied from3.3 × 10 −3/s to6.3 × 10 −3/s. The variations in each parameter were not statistically significant at any age. These data represent the first characterization of diffusion parameters in a developing brain. They confirm previous histological indications of a relatively large extracellular volume fraction during early postnatal development. The constancy of the tortuosity shows that diffusion of small molecules is no more hindered in the developing brain than in the adult. The large extracellular space volume fraction of the neonatal brain could significantly dilute ions, metabolites and neuroactive substances released from cells, relative to release in adults, and may be a factor in preventing anoxia, seizure and spreading depression in young animals. The diffusion characteristics could also play an important role in the developmental process itself.

First appearance, distribution, and origin of macrophages in the early development of the avian central nervous system

A phagocytic cell system of hemopoietic origin exists in the early avian embryo (Cuadros, Coltey, Nieto, and Martin: Development 115:157-168, '92). In this study we investigated the presence of cells belonging to this system in the central nervous system (CNS) of chick and quail embryos by using both histochemical staining for acid phosphatase and immunolabelling with antibodies recognizing cells of quail hemangioblastic lineage. The origin of these cells was traced in interspecific chick-quail yolk sac chimeras. Hemopoietic cells were detected within the CNS from developmental stage HH15 on, and steadily increased in number at subsequent stages. Analysis of yolk sac chimeras revealed that most of these cells were of yolk sac origin, although some hemopoietic cells of intramebryonic origin were also found in the CNS. Immunocytochemical, histochemical, and ultrastructural characterization allowed us to identify hemopoietic cells in the CNS as macrophages. These cells were consistently found in the brain vesicles and spinal cord, appearing (1) between undifferentiated neuroepithelial cells at dorsal levels of the CNS; (2) in areas of cell death; (3) in the marginal layer in close relationship with developing axons; (4) in large extracellular spaces in the subventricular layer; (5) on vascular buds growing through the marginal and subventricular layers; and (6) in the ventricular lumen. Macrophages in different locations varied in morphology and ultrastructure, suggesting that in addition to their involvement in phagocytosis, they play a role in other processes in the developing CNS, such as axonal growth and vascular development. The first macrophages migrate to the CNS independently of its vascularization, apparently traversing the pial basal lamina to reach the nervous parenchyma. Other macrophages may enter the CNS together with vascular buds at subsequent stages during CNS vascularization.

Ion concentrations, fluxes and electrical properties of the embryonic chicken lens

The membrane properties of embryonic chicken lenses were characterized using isotopic and electrical techniques. The lenses had a relatively high water content (80%) and large extracellular space (12·5%). Isotopic uptake measurements indicated that the lens cytoplasm contained 118 m m K + and 26 m m Cl −. A value for intracellular Na + of 14 m m was obtained using Na +-sensitive microelectrodes. A doubleexponential model was used to fit the efflux of 86Rb +, 22Na +, 36Cl − and [ 3H]mannitol (an extracellular space marker) from the lens. When perfused with artificial aqueous humor (AAH) solution, embryonic lenses exhibited membrane potentials of between −20 and −40 mV. The more negative values were generally observed in lenses from older embryos. A ouabain-sensitive component, contributing −7 mV to the membrane potential, was also identified. The relatively depolarized membrane potentials suggested that the lens membranes were only weakly selective for K + over Na +. To test this further, lenses were perfused with AAH containing varying concentrations of K +. The resulting changes in potential were interpreted in terms of the Goldman model. The best fit of the Goldman potential equation indicated that, in the presence of ouabain, the chicken lens membranes had a relative permeability to K +, Na + and Cl − of 1·0, 0·36, 0·51 respectively. Replacing most or all of the Na + in the AAH caused only a small change in the membrane potential rather than the large hyperpolarization towards the K + equilibrium potential predicted by the Goldman model. Including the K + ionophore valinomycin in the low Na +-AAH solutions caused a large increase in 86Rb + efflux but did not result in additional hyperpolarization. This suggested that the insensitivity of the membrane potential to reduced extracellular Na + was not due to voltage or pH inactivation of lens K + channels.

Characteristics of activity-dependent potassium accumulation in mammalian peripheral nerve in vitro

Ion-sensitive microelectrodes were used to study the behavior of extracellular ions in rat sciatic nerve during and following activity. Nerve stimulation produced increases in [K +] o that were dependent upon the frequency and duration of stimulation; no change in extracellular pH occurred with stimulation. Increases in [K +] o dependent on axonal discharge since they were blocked by inhibiting sodium channels with tetrodotoxin. At 22°C, stimulation could induce increases in [K +] o of several mM; at 36°C, stimulation rarely produced increases in [K +] o greater than 1mM. Stimulated increases in [K +] o dissipated very slowly (i.e. t 1/2 = 50–100s) and the rate of dissipation was not significantly affected by anoxia, changes in temperature, changes in extracellular pH, or the application of a blocker of Na +, K +-ATPase (ouabain) or a K + channel blocker (Ba 2+). In comparison to the central nervous system, neural activity in rat sciatic nerve produced smaller increases in [K +] o and these increases dissipated much more slowly. The primary mechanism of K + dissipation appeared to be diffusion, probably facilitated by the larger extracellular space in peripheral nerve compared to the central nervous system, but impeded by diffusion barriers imposed by the blood-nerve barrier.

Electron microscopic and immunocytochemical study of rapidly frozen, freeze-substituted neural lobes of rats

Rapid freezing of freshly dissected or incubated neural lobes was explored as a means of obtaining ultrastructural preservation of the more natural state of this tissue. A quantitative assessment of the region of good fixation was made in order to determine the relative fractions occupied by axons, pituicytes and the extracellular space. The immunocytochemical distributions of neurophysins and the glycopeptide portion of the vasopressin precursor were evaluated using the immunogold technique in order to determine the relative numbers of oxytocin and vasopressin fibre types in the fixed region, and the subcellular localization of these antigens. The uncut surface of rat neural lobes was rapidly frozen against a highly polished copper plug and freeze-substituted in osmium-acetone either immediately after dissection (approximately 2 min), or after a 15 min incubation period in vitro in an oxygenated, balanced salt solution. Substituted neural lobes were prepared for either conventional electron microscopy, or for immunogold labelling of neurophysins and the glycopeptide precursor to vasopressin. Membranes, subcellular organelles and extracellular matrix were well preserved 10 microns deep to the contacted surface. The extracellular space accounted for approximately 30% of the cross-sectional area of the neuropil and could be divided into two domains: an extended perivascular space (28-29% of total area); and a narrow (approximately 24 nm; approximately 1% of total) space between closely apposed neurosecretory processes or between these processes and pituicytes. Pituicytes accounted for about 30% of the area and axons 20-25%. Pituicytes occupied close to 60% of the basal lamina at the neurohaemal contact zone, while axons occupied approximately 20%. There were no differences between neural lobes frozen immediately after dissection and those incubated for 15 min in any of these measures, suggesting minimal fluid redistribution. Gold particles were specifically localized over large (100-200 nm) dense core vesicles, and less frequently over multivesicular bodies and lysosomes. No etching of the plastic or reduction of osmium was necessary to achieve labelling. Specific labelling of one set of terminals and axons (about 80%) was observed with the monoclonal antibody previously shown to be specific for oxytocin-neurophysin, while in neighbouring sections the remaining 20% of the processes were labelled with the antiserum to the vasopressin precursor, or with non-specific antibodies to neurophysins. In conclusion, ultrarapid freezing preserves a large extracellular space in the neural lobe and provides for high resolution morphological and immunocytochemical studies of neurohypophysial structure.

Molecular and cellular characterization of the glial roof plate of the spinal cord and optic tectum: A possible role for a proteoglycan in the development of an axon barrier

Certain types of glial structures, located at strategic positions along axon pathways, may provide the mechanical and/or chemical elements for the construction of barriers which can grossly direct the elongation of axons during development. The roof plate, a putative axon barrier, is located along the dorsal midline of the developing spinal cord and may be important for the guidance of the commissural and dorsal column axons. We examined the roof plate to determine the developmental morphology of the region and to determine which molecules were correlated with the barrier function when axons were growing nearby. Light and electron microscopic observations of the roof plate revealed that this glial domain undergoes a dramatic change in shape from a “wedge” with large extracellular spaces between the cell apices at E12.5 to a thin, dense septum with reduced extracellular space at E15.5. Immunocytochemical techniques demonstrated that highly sialylated neural cell adhesion molecule (N-CAM), the carbohydrate recognized by L2 monoclonal antibody, cholinesterase, stage-specific embryonic antigen 1, and a ligand that binds tetragonolobus purpureas agglutinin are expressed by the roof plate. These molecules, however, were also found in other regions of the spinal cord which are permissive or attractive to axon growth. A molecule which is unique to the roof plate when axons grow close to, but do not cross, the dorsal midline is a glycosaminoglycan (GAG), keratan sulfate. Keratan sulfate is also present in the tectal midline and in other noninnervated regions such as the outer epidermis and developing cartilage. Our data suggest that keratan sulfate, alone or in combination with other molecules expressed by the roof plate, may be responsible, in part, for the inhibition of axon elongation through the roof plate in the embryonic spinal cord.

First month of development of fetal neurons transplanted as a cell suspension into the adult CNS

It has been demonstrated elsewhere that fetal thalamic tissue, when transplanted as a cell suspension into the excitotoxically neuron-depleted adult somatosensory thalamus, can grow, differentiate, and receive projections from host afferents. In the present study, we used the same paradigm to analyse the transplanted neurons during their morphogenesis, i.e. during the first month after transplantation. Using various anatomical criteria, at the light and electron microscope levels, we compared the development of transplanted neurons with the normal ontogeny of homologous neuronal populations. Confined solely to the mechanically lesioned area during implantation at seven days post-grafting, the transplant increased in size to occupy most of the previously neuron-depleted area by the third week after grafting. The final size of the transplant thus depended upon the size of the lesion. At seven days post-grafting, the neurons were small in size and the cellular density was high. At this immature stage few synaptic contacts were visible and the ultrastructure was characterized by large extracellular spaces. At 10 days post-grafting, the size of the neurons had increased and the cellular density had decreased. Both an extensive dendritic proliferation and a simultaneous active synaptogenesis could also be observed. All these events continued to evolve and during the third week the neuropil progressively acquired more mature ultrastructural characteristics. Synaptic contacts exhibiting characteristics comparable to those observed in the intact thalamus also became more numerous. At 20 days post-grafting, axonal myelination had started, the development of the graft apparently stopped and the various criteria had stabilized. Until that developmental stage, growth of grafted neurons compared to that of normal thalamic ones. At later stages, however, grafted neurons failed to grow larger and did not reach the size of the homologous population in the adult animal. It seems, therefore, that transplants of thalamic fetal neurons can be used as a tool with which to study thalamic neuronal development, within definable limits.

Cell death in suboptic necrotic centers of chick embryo diencephalon and their topographic relationship with the earliest optic fiber fascicles.

The structural features of suboptic necrotic centers (SONCs) in the floor of the chick embryo diencephalon were studied. These necrotic areas were observed lateral to the prospective zone of the optic chiasm through developmental stages 14 to 24. The relationship between SONCs and the earliest optic fiber fascicles also was studied in an attempt to determine the possible significance of these cell death areas in the mechanism of optic pathway development. In SONCs, healthy neuroepithelial cells contain primary lysosomes and phagocytose fragments of dead cells. Discrete regions within the cytoplasm of some cells show electron-transparent vacuoles in contact with dense contents of ruptured lytic bodies. The cytoplasm of dying cells and dead cell fragments are notably electron dense, with numerous secondary lysosomes and electron-transparent vacuoles. These observations are interpreted on the assumption that after autophagic processes, condensation and fragmentation take place in dying cells of the SONCs. In the ventricular lumen adjacent to the SONCs, numerous more or less spherical bodies are observed that appear to be shed from the tip of the cells constituting the SONCs. Three different types of intraventricular bodies can be distinguished: loose, moderately dense, and highly dense. The first type appears to originate from apical portions of cells that undergo autolytic processes. Moderately dense fragments are interpreted as originating from dying cells in which the cytoplasm is undergoing condensation. Finally, highly dense intraventricular bodies appear to be fragments of dead cells that are shed into the ventricular lumen. SONCs separate the prospective area of the optic chiasm from lateral regions of the diencephalic floor. Extracellular spaces are poorly developed within the wall of the SONCs, whereas the neuroepithelium of the presumptive optic chiasm and regions located rostral and caudal to SONCs show abundant and extensive extracellular spaces. These are bounded by long marginal processes of neuroepithelial cells. Sagittal sections of embryonic heads at stages 22-24 reveal optic fiber fascicles penetrating the SONCs asymmetrically, as they are found only in its caudal half. These observations suggest that the SONCs function as doorways made of compact neuroepithelium, to be traversed by the earliest optic fibers before they reach the middle zone of the floor of the diencephalon through which they travel to the contralateral optic tract within large extracellular spaces.

Axon pathway boundaries in the developing brain. I. Cellular and molecular determinants that separate the optic and olfactory projections

When optic fibers first approach the chiasmatic region of the diencephalon in the chick embryo on days 3 and 4 (E3-4), they rarely grow rostrally into the olfactory region of the telencephalon. Conversely, olfactory tract axons grow as far as, but never cross the diencephalic/telencephalic (D/T) boundary to enter the optic chiasm. In this study, a region of specialized neuroepithelium, originally named the "knot" in mouse by Silver (1984), has been identified at the D/T border of chick embryos. At pre-axonal stages, the presumptive knot region undergoes a cataclysmic cell death, with concomitant phagocytosis of necrotic debris by the remaining cells. When fibers subsequently appear in the chiasm and olfactory tracts, the knot consists of a very dense, interwoven cluster of non-neuronal cells that lack marginal radial processes, and whose cell bodies directly abut the glial limiting membrane. Thus, the morphology of the knot is in sharp contrast to the cell body-free marginal zone and endfoot regions along which axons tend to grow. In addition, we found that the neural cell adhesion molecule (N-CAM), which is expressed on neuroepithelial cell processes within the central optic and olfactory pathways, is not present on cells in the knot region during periods of axon growth. These results suggest that the knot, through its elimination of the marginal zone processes, absence of large extracellular spaces, and relative absence of adhesion molecules, functions as an axon-refractory barrier that effectively separates the optic and olfactory projections.

Extra-axonal environment and fibre directionality in the early development of the chick embryo optic chiasm: a light and scanning electron microscopic study.

The events that occur during the early development of the optic chiasm of the chick embryo have been studied by light and scanning electron microscopy. In developmental stages previous to the arrival of the first optic fibres in the floor of the diencephalon, as well as during the arrival of the leading fibres, extracellular spaces can be seen in the diencephalon ventral wall. These spaces are defined by external cell prolongations which end in a foot-shaped formation. During stages 25 and 26 a prechiasmic degenerative centre appears in the area immediately rostral to the early chiasm, leading to a notable degree of disorganization in the diencephalon wall. This centre appears to be related to the reorganization of the system of external cell processes and extracellular spaces which become progressively more irregularly distributed, coinciding with the arrival of the first optic fibre fascicles to the midline of the floor of the diencephalon. The optic fibre fascicles change their latero-medial directionality in the medial-most regions of the ventral diencephalon, where their course becomes rostrocaudal. This reorientation of the optic fibres seems to be mediated by primitive glial cells which first appear in the ventrorostral region of the early chiasm (previously occupied by the system of external cell processes and extracellular spaces) in stage 26, increasing in number from this stage on. The morphology of the primitive glial cells is laminar in nature and the cells are seen to be densely packed together with no large extracellular spaces between them.